Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding
Amphibians as a model to study endocrine disruptors: I. Environmental pollution and estrogen receptor binding
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DOI：
10.1016/s0048-9697(99)80016-3
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发表时间：
1999-01-12
影响因子：
9.8
通讯作者：
Kloas, W
中科院分区：
文献类型：
--
作者：
Lutz, I;Kloas, W
许多释放到环境中且没有毒理学风险的化学物质具有破坏内分泌系统功能的能力。这些内分泌干扰物会扰乱正常的内分泌机制，并且在几乎所有类别的脊椎动物中都观察到。这项研究的目的是开发一个综合模型，利用两栖动物非洲爪蟾研究内分泌干扰。内分泌干扰物的雌激素效力评估包括几个层面的研究：（I）与肝脏雌激素受体的结合，（II）通过诱导原代培养的肝细胞中卵黄蛋白原合成而产生的体外雌激素活性，以及（III）幼虫暴露引起的体内对性发育的影响。本文的重点是第一部分，通过使用肝胞质级分建立 [H-3]17 β-雌二醇 ([H-3]E2) 结合的放射受体测定法。为了获得最佳的结合条件，我们进行了动力学、饱和和竞争性位移实验。 [H-3]E2 与雌激素受体的结合表明，在孵育 18 至 48 小时之间实现最大特异性结合。饱和实验的斯卡查德分析产生了同质的可饱和雌激素受体群体，两性之间的结合参数没有显着差异。男性和女性的 K-d（解离常数）值分别为 22.4 +/- 6.0 和 15.0 +/- 2.8 nM（平均值 +/- S.E.M.；n = 5），而相应的 B-max（最大结合能力）显示为 89 +/- 46 和 136 +/- 46 fmol [H-3]E2/mg 蛋白。竞争性置换实验显示的雌激素受体的特异性证明受体仅对雌激素具有高度特异性，但对具有以下结合亲和力排名的其他内源性类固醇不具有高度特异性：E2>雌酮>脱氢表雄酮>醛固酮大于或等于睾酮大于或等于皮质酮大于或等于黄体酮。与E2相比，环境化学品的亲和力排名为：E2>四氯联苯>邻苯二甲酸二乙酯>2,2-双-(4-羟基苯基)-丙烷(双酚A)大于或等于4-壬基苯酚大于或等于3-叔丁基-4-羟基茴香大于或等于4-辛基苯酚>二氯二苯三氯乙烷(4,4'-DDT)。对五种污水废水中 [H-3]E2 结合的置换进行分析，结果发现三个样品置换了原始浓度下超过 50% 的特异性结合。总而言之，已建立的非洲爪蟾肝细胞质中[H-3]E2结合的放射受体测定可用于筛选纯化合物或它们的复杂混合物的雌激素受体结合，这是引起雌激素或抗雌激素作用的先决条件。 (C) 1999 Elsevier Science B.V. 保留所有权利。
Many chemicals released into the environment without toxicological risks have the capacities to disrupt the function of endocrine systems. These endocrine disrupters disturb normal endocrine mechanisms and have been observed in nearly all classes of vertebrates. The aim of this research is to develop a comprehensive model to study endocrine disruption using the amphibian Xenopus laevis. The assessment of estrogenic potencies of endocrine disrupters includes several levels of investigation: (I) binding to liver estrogen receptor, (II) estrogenic activity in vitro by inducing vitellogenin synthesis in primary cultured hepatocytes, and (III) in vivo effects on sexual development caused by exposure of larvae. The present paper is focused on the first part by establishing a radioreceptorassay for [H-3]17 beta-estradiol ([H-3]E2) binding using liver cytosol fraction. In order to get optimum binding conditions we performed kinetic, saturation, and competitive displacement experiments. Association of [H-3]E2 to estrogen receptor revealed that maximum specific binding is achieved between 18 and 48 h of incubation. Scatchard analyses of saturation experiments resulted in a homogenous saturable population of estrogen receptors having no significant differences of binding parameters between both sexes. The values of K-d (dissociation constant) in males and females were 22.4 +/- 6.0 and 15.0 +/- 2.8 nM (mean +/- S.E.M.; n = 5), respectively, while corresponding B-max (maximum binding capacity) revealed 89 +/- 46 and 136 +/- 46 fmol [H-3]E2/mg protein. The specificity of estrogen receptors as shown by competitive displacement experiments demonstrated receptors being highly specific just for estrogens, but not for other endogenous steroids having the following ranking of binding affinities: E2 > estrone > dehydroepiandrosterone > aldosterone greater than or equal to testosterone greater than or equal to corticosterone greater than or equal to progesterone. The affinity ranking of environmental chemicals compared to E2 was: E2 > tetrachlorbiphenyl > diethylphthalate > 2,2-bis-(4-hydroxyphenyl)-propan (bisphenol A)greater than or equal to 4-nonylphenol greater than or equal to 3-t-butyl-4-hydroxyanisale greater than or equal to 4-octylphenol > dichlor-diphenyl-trichlor-ethan (4,4'-DDT). Analyses of five sewage effluents for displacement of [H-3]E2 binding resulted in three samples displacing more than 50% of specific binding at their original concentration. Taken together the established radioreceptorassay for [H-3]E2 binding in Xenopus laevis liver cytosol is useful to screen estrogen receptor binding of pure compounds or complex mixtures of them, which is the prerequisite for causing either estrogenic or antiestrogenic effects. (C) 1999 Elsevier Science B.V. All rights reserved.